Otitis media (OM) is the most common bacterial infection in the United States among young children for which medical care is sought, and Streptococcus pneumoniae strains are the leading cause of bacterial OM. These gram-positive diplococci colonize the respiratory tract in up to 50% of healthy children and only cause disease under special circumstances. Recent genomic sequencing projects and advancements in molecular biology have resulted in the identification of numerous putative virulence factors. These factors have mainly been studied in the context of invasive pneumococcal diseases such as pneumonia, or have been studied in one or a few laboratory strains. While more virulence factors remain to be discovered, the new challenge is to identify which of these many factors warrant further study, to link these factors specifically to OM pathogenesis, and to estimate the relative importance of these virulence factors among the S. pneumoniae strains in circulation. This project is built on the observation that S. pneumoniae strains differ in their ability to cause disease, and that these differences are likely due to genetic differences between strains that extend beyond the polysaccharide capsule. The goal of this project is to identify genes associated with pneumococcal OM and to evaluate the relative frequency of pneumococcal virulence genes among a collection of isolates obtained from healthy children and children with clinical disease. A four step interdisciplinary approach utilizing techniques of molecular biology and epidemiology will include: 1. Selection of S. pneumoniae strains for genomic subtraction with the highest potential to identify genes associated with OM. 2. Identification of DNA sequences (sPCR fragments) unique to strains causing OM (tester strains) and absent in strains from healthy carriers (driver strains) using genomic subtraction. 3. Epidemiologic screening of a large collection of isolates from healthy children and children with OM, meningitis, pneumonia, or bacteremia using sPCR fragments. 4. Identification of genes associated with sPCR fragments important for OM pathogenesis and description of their biological and clinical characteristics. Discovery of additional factors involved in streptococcal OM will facilitate the development of new strategies for the control and prevention of this important disease.